Enzymatic determination of nitrogen-
containing compounds and enzymes
reactive therewith

ABSTRACT

A PROCESS FOR MEASURING THE AMOUNT OF A NITROGENCONTAINING COMPOUND IN A SAMPLE OR THE AMOUNT OF AN ENZYME IN A SAMPLE, THE ENZYME BEING REACTIVE WITH A NITROGEN-CONTAINING COMPOUND, WHICH INVOLVES (1) MIXING THE SAMPLE WITH A COMPOUND THAT IS ENZYMATICALLY REACTIVE WITH AMMONIA; (2) ADDING TO THE NITROGENCONTAINING COMPOUND IN THE SAMPLE AN AMOUNT OF THE ENZYME OR ADDING TO THE ENZYME A NON-RATE LIMITING AMOUNT OF A NITROGEN-CONTAINING COMPOUND TO RELEASE AMMONIA, IN EITHER CASE, AT A MEASURABLE RATE; AND (3) MEASURING THE RATE OF REACTION OF AMMONIA WITH THE COMPOUND THAT IS REACTIVE WITH AMMONIA, THIS RATE OF REACTION BEING PROPORTIONAL TO THE CONCENTRATION OF THE NITROGEN-CONTAINING COMPOUND OR THE CONCENTRATION OF THE ENZYME, AS THE CASE MAY BE, IN THE ORIGINAL SAMPLE.

United States Patent Oflice Re. 27,524 Reissued Nov. 28, 1972 Matterenclosed in heavy brackets If] appears in the original patent but formsno part of this reissue specification; matter printed in italicsindicates the additions made by reissue.

ABSTRACT on THE DISCLOSURE A process for measuring the amount of anitrogencontaining compound in a sample or the amount of an enzyme in asample, the enzyme being reactive witha nitrogen-containing compound,which involves (1) mixing the sample with a compound that isenzymatically reactive with ammonia; (2)v adding to thenitrogencontaining compound in the sample an amount ;of the enzyme oradding to the enzyme a non-rate limiting amount of a nitrogen-containingcompound to release ammonia, in either case, at a measurable rate; and

(3) measuring the rate of reaction of ammonia with the compound that isreactive with ammonia, this rate of reaction being proportional to theconcentration of the nitrogen-containing compound or the concentrationof the enzyme, as the case may be, in the original sample.

This invention relates to a procedure for the quantitative determinationof nitrogen in nitrogen-containing compounds and, more particularly, toa combination of enzymatic reactions for such determination.

While the invention will be described specifically for the determinationof the amount of urea in blood samples, urine samples, etc., theinvention is not so limited. The invention is applicable whereverammonia can be produced from a parent nitrogen-containing compound by anenzymatic reaction at a rate or inan amount related to the amount orconcentration of the parent compound.

Urea, the diamide of carbonic acid, is the most important product ofprotein metabolism in humans and other mammals and in various otheranimal species.

There are many methods foriden'tifying and determining urea. One suchmethod involves hydrolyzing urea with the enzyme urease to producecarbon dioxide and ammoniaand then determining either the carbon dioxidegasometrically or the ammonia colorimetrically with Nesslers reactionwithout prior deproteinization. It is a further object to provide aprocedure for nitrogen determination that is specific, simple, rapid andsensitive. Other objects will appear hereinafter.

The objects are accomplished by a process that, in its broadest terms,couples an ammonia producing enzyme reaction directly and simultaneouslywith a second reaction which enzymatically incorporates ammonia as fastas the first reaction produces the ammonia. The basic requirement forthe first reaction is that this reaction produce ammonia at a rate or inan amount that is proportional to the parent compound to be determined,e.g., urea, l-lysine, glycine, etc. The requirements for the secondreaction are: (1) it must be compatible with the first reaction; (2) itmust be specific for the ammonia released by the first reaction; (3) therate of the second reaction must be limited only by the rate of ammoniaformation from the first reaction; and (4) the rate of the secondreaction must be susceptible to being read by suitable means, e.g.,spectrophotometric procedures, colorimetric procedures, fiuorometricprocedures, turbidimetric procedures, conductometric procedures,amperometric procedures, etc.

Specifically, a process for determining urea comprises .the steps, insequence, of adding a measured amount of the urea-containing sample to asolution containing the reduced form of fi-nicotinamide-adeuinedinucleotide, sodium-a-ketoglutarate and l-glutamic dehydrogenase, ,thesolution being buffered to a pH of about 6.8 to about 9.0, preferablyfrom 7.8 to 7.9; and, thereafter, adding an aqueous solution of ureaseand measuring the rate of formation of oxidized fi-nicotinamide-adeninedinucleotide. f.

Since the reduced form of fi-nicotinamide-adenine dinucleotide absorbslight very strongly at 340 millimicrons while the oxidized form doesnot, the rate of disappearance of the reduced formis directlyproportional to the decrease in absorbance of light at 340 millimicronsat constant temperature, usually. at a constant temperature between 15C. and 50 C., and can be measured readily by those skilled in the artusing a conventional spectrophotometric procedure. Since the rate of.oxidation of the reduced form of fl-nicotinamide-adenine dinucleotideis also proportionalto the rate of formation of ammoniaand the rate offormation of ammonia is a function of the concentration of the parentnitrogencont aining compound, the decrease in absorbance of 340millimicrons can be used as a direct measure of the originalconcentration of the parent nitrogen-containing compound. It should alsobe understood that other wavelengths, e.g., 366 millimicrons, can beusedfor the foregoing purpose.

Although sodium a-ketoglutarate has been designated for use in thepreferred process of the invention, it should be understood that othersalts of the a-ketoglutaric acid or the acid may be used. The onlylimitation is that .such salt or any acid present must be dissolved in,and compatible with, the reaction system. Thus, by specifyinga-ketoglutarate, it is meant that such salts as the sodium, potassium,etc. salt of u-ketoglutaric acid are used.

As set forth previously, this same procedure can be used for thequantitative determination of nitrogen in other parent compounds thatyield, ammonia, besides urea.-In the following table, Table I, areprovided parent nitrogen-containing compounds that can be determined bythe procedure of this invention and the enzymes to be used in theammonia-releasing reaction of the procedure. After determining theoptimum conditions of temperature, pH, ionic strength, etc. for securinga pseudo zero order kinetic condition, Le, a linear portion in the curveobtained by plotting the rate of change in absorbance versus nitrogenconcentration, the procedures for all the determinations becomeidentical and will be more readily understood by reference to theexample presented hereinafter.

TABLE I Parent compound Enzyme l-Lysine l-Lys'ine amino acid oxidase.l-Hist-idine Histidine-a-deaminase. l-Serine l-Serinel dehydrase.l-Threonine l-Threonine amino acid ox-idase. l-Homoserine l-Hornoserineamino acid oxidase. l-Cysteine l-Cysteine amino acid oxidase. GlycineGlycine oxidase. l-Asp'artic acid l-Asparta-se. Quanine Quan inedeam'in-ase.

Aliphatic amines Monoamine oxidase. d-Amino acids d-Arnino acidox'idase.

l-Glutamine l-G'lut-aminase.

Uric acid Ur-icase.

It is apparent that the basic ideas involved in the procedures of thisinvention can also be used to determine the amount of enzyme present.For such a procedure, the parent nitrogen-containing compounds (urea,l-lysine, etc.) would be added in the non-rate limiting concentrationrather than the enzyme (urease, l-lysine amino acid oxidase, etc.). Themeasurement of the rate of oxidation of the reduced form offi-nicotinamideadenine dinucleotide would then be a function of theoriginal enzyme concentration.

It will be observed that in the so-called second reaction, theammonia-incorporating reaction, the ammonia reacts withsodium-m-ketoglutarate to form l-glutamate while the reduced form of thenicotinamide-adenine dinucleotide is oxidized. Alternatively, theammonia can be incorporated into pyruvic acid by using the enzymel-alaninedehydrogenase (isolated from Bacillus subtilis). This reactionin which ammonia is incorporated into pyruvic acid also requires the useof the reduced form of fl-nicotinamide-adenine dinucleotide which isoxidized during the reaction. The oxidation of the B-nicotinamideadeninedinucleotide can then be determined in the spectrophotometric mannerpreviously described, by the change in absorbance at 340 millimicrons,to provide a quantitative determination of the nitrogen-containingparent compound. Still another alternative reaction for the detection ofammonia is the reductive incorporation of ammonia into glyoxylate by theenzyme glyoxylate reductase which can be obtained from Microbacteriumtuberculosis, Hominis. The rate of this reaction can also be readilydetermined by the decrease in absorbance at 340 millimicrons, since thisenzyme also requires reduced p-nicotinamide-adenine dinucleotide as thecoenzyme.

The procedure described which involves a coupled reaction system andwhich can be used for determining amounts of parent nitrogen compoundsas well as for the specific assay of enzymes has many advantages. It isa procedure that is specific; the enzymes have the ability to reactspecifically with the individual components of complex mixtures. Sincethe procedure is extremely specific, the necessity for thetime-consuming separation of components is minimized. Furthermore, theprocedure does not require the complete conversion of the compo nents tobe assayed. Thus, the complex conversion of the specific componentsassayed can be accomplished in a very short time. It will be also notedthat the conditions employed in the reactions are sufficiently mild toallow the detection and determination of labile substances. Finally, theprocedure is extremely sensitive; it will operate where concentrationsof the parent nitrogen-containing compound or the enzyme, whichever isto be determined,

4 are extremely low, e.g., as low as 0.2 microgram per milliliter ofassay solution. Other advantages will be apparent from the followingexample.

EXAMPLE This is an example for determining the urea nitrogenconcentration in samples of blood serum or blood plasma. If urine orother ammonia containing materials are used, the sample should be passedthrough an ion exchange column, or other suitable means should be used,prior to analysis in order to remove free ammonia.

The procedure depends upon the following two reactions:

Urease (1) Urea-l-water ammonia-l-carbon dioxide (2)Ammonla+a-ketoglutrate+the reduce form of fl-niootinamldeadeninedinucleotide oxidized Zinc ion Bnieotinamide-adeninedinucleotide+l-glutamate+water.

The first series of runs serves to provide a relationship between therate of the reaction occurring in Equation 2 and known amounts of ureanitrogen in standard solutions. For these runs, 4.96 milliliters of areagent solution for carrying out the second reaction are pipetted intoa one-centimeter optical cell; the cell is placed in a water bath at 37C. for 3 minutes; and 0.020 milliliter of the solution to be evaluatedis added.

The reagent solution has been prepared by dissolving 34.2 milligrams ofl-cysteine hydrochloride in 25 milliliters of a phosphate butter. Thebuffer is prepared by mixing 84 milliliters of 1.0 molar sodiumdibasicphosphate and 16 milliliters of 1.0 molar potassiummonobasicphosphate, diluting the resulting mixture to 1 liter withdistilled water, and then adjusting the pH to 7.85. 2.4 milliliters of asolution of 0.1 gram of the disodium salt of fl-nicotinamide-adeninedinucleotide in 10 milliliters of the phosphate buffer; 2.4 millilitersof a solution of sodium-a-ketoglutarate prepared by dissolving 0.885gram of the monosodium salt of a-ketoglutaric acid in 2 milliliters ofwater and adjusting the pH to 7.8 with 1.0 normal sodium hydroxide andadding sufficient phosphate buffer to bring the volume to 25milliliters; and 5 milliliters of ammonia-free l-glutamic dehydrogenasesuspended in a solution of 5 0% glycerol and 0.1 molar phosphate bufferat a pH of 7.0, the suspension containing 30 units of the enzyme permilliliter, are all added to the l-cysteine hydrochloride solution; andthe solution is diluted with the phosphate buffer to a total volume of100 milliliters.

0.02 milliliter of various standard solutions containing known amountsof urea nitrogen are then added to provide the reference curve. Theseamounts range from 200 milligrams of nitrogen per 100 milliliters ofphosphate buffer (200 mg. percent), prepared using 2.148 grams of ureain 500 milliliters of phosphate bulfer, down to 10 milligrams ofnitrogen per 100 milliliters of phosphate buffer (10 mg. percent),prepared by diluting 5 milliliters of the first prepared urea solutionwith milliliters of the butter.

After a two-minute delay following the addition of the test solution,0.02 milliliter of a solution of 10 milligrams of urease (2500units/gram) in 1 milliliter of a 50% glycerol-distilled water solutionis added to the optical cell. While maintaining the temperature of theoptical cell at 37 C., absorbance versus time at a wavelength of 340millimicrons is read using a spectrophotometer. From the linear portion(usually from 30 seconds to 2 minutes after the urease addition) thereaction rate (A absorb ance/minute) is obtained for each known ureanitrogen concentration and the results are plotted as the standardreference curve.

After preparing the standard reference curve, the foregoing procedure isrepeated but substituting a 0.02 milliliter sampleof the blood serum orblood plasma, etc. for the standard solutions of urea nitrogen. Theabsorbance at a wavelength of 340 millicrons is read and recorded as afunction of time. From the reaction rate obtained in the linear portionof this curve (usually from 30 seconds to 2 minutes after the ureaseaddition), the urea nitrogen is read oil the standard reference curve.

What is claimed is:

1. A process for the quantative determination of a nitrogen-containingcompound capable of releasing ammonia upon enzymatic action whichcomprises adding a measured amount of a sample of suchnitrogen-containing compound to a solution of non-rate limiting amountsof a first enzyme and compound A, said compound A being reactive withammonia in a reproducible measurable manner in the presence of saidfirst enzyme; thereafter, adding a second enzyme that reacts with saidnitrogencontaining compound to release ammonia, the amount of saidsecond enzyme added being sufiicient to release ammonia from said sampleat a measurable rate proportional to the concentration of thenitrogen-containing compound ,in said sample; and measuring the rate ofreaction of ammonia with said compound A while maintaining the system ata substantially constant temperature and at a substantially constant pH.

2. A process for the quantitative determination, of anitrogen-containing compound capable of releasing ammonia upon enzymaticaction which comprises adding a measured amountof a sample'of suchnitrogen-containing compound to a solution of non-rate limiting amountsof l-glutamic dehydrogenase, a-ketoglutarate, and the "reduced form offl-nicotinamide-adenine dinucleotide; thereafter, adding a second enzymethat reacts with said nitrogen-containing compound to release ammonia,the amount of said second enzyme added being sufiicient to releaseammonia from said sample at a measurable rate proportional to theconcentration 'of the nitrogen-containing compound in said samplefandmeasuring the rate of disappearance of the reduced form of[3-nicotinamideadenine dinucleotide while maintaining the system at asubstantially constant temperature and at a substantially constant pH.

, 3. A process as in claim 2 wherein said nitrogen-containing compoundisselected from the group consisting ,of urea, uric acid, l-lysine,l-histidine, l-serine, l-threonine,

l-homoserine, l-cysteine, glycine, l-aspartic acid, quanine, aliphaticamines, d-amino acids and l-glutamine, and said enzyme is,correspondingly, selected from the group consisting of urease, uricase,l-lysine amino acid oxidase,

histidine a deaminase, l-serine dehydrase, l-threonine amino acidoxidase, l-homoserine amino acid oxidase, lcysteine amino acid oxidase,glycine oxidase, l-aspartase, quaninedeaminase, monoamine oxidase,d-amino acid ox- ,idase and l-glutaminase.

4. A process forthe quantitative determination of a nitrogen-containingcompound capable of releasing ammonia upon enzymatic action whichcomp-rises adding a "measured amount of a sample of suchnitrogen-containing compound to a solution of non-rate limiting amountsof l-alanine dehydrogenase, pyruvate, and the reduced form offl-nicotinamide adenine dinucleotide; thereafter, add- .ing a secondenzyme that reacts with said nitrogen-containing compound to releaseammonia, the amount of said second enzyme added being sufiicient torelease ammonia from said sample at a measurable rate proportional tothe concentration of the nitrogen-containing compound in ,said sample;and measuring the rateof disappearance of the reduced form offl-nicotinamide-adenine dinucleotide while maintaining the system at asubstantially constant temperature and at a substantially constant pH.

5. A process for the quantitative determination of anitrogen-containing.compound capable of releasing ammonia upon enzymaticaction which comprises adding a measured amount of a sample of suchnitrogen-containing compound to a solution of non-rate limiting amountsof glyoxylate reductase, glyoxylate, and the reduced form ofti-nicotinamide-adenine dinucleotide; thereafter, adding a second enzymethat reacts with said nitrogen-containing compound to release ammonia,the amount of said second enzyme added being sufiicient to releaseammonia from said sample at a measurable rate proportional to theconcentration of the nitrogen-containing compound in said sample; andmeasuring the rate of disappearance of the reduced form ofB-nicotinamide-adenine dinucleotide while maintaining the system at asubstantially constant temperature and at a substantially constant pH.

6. A process for the quantitative determination of urea which comprisesadding a measured amount of a ureacontaining sample to a solution ofnon-rate limiting amounts of l-glutamic dehydrogenase, a-ketoglutarateand the reduced form of fl-nic'otinamide-adenine dinucleotide, saidsolution being buffered to a pH of 6.8-9; thereafter,

adding an amount of a solution of urease suflicient to 1 release ammoniafrom said urea-containing sample at a measurable rate proportional tothe concentration of urea in said sample; and measuring the rate ofdisappearance of the reduced form of fl-nicotinamide-adeninedinucleotide while maintaining the system at a substantially constanttemperature selected from between 15 C. and 50 C.

7. A process for the quantitative determination of urea which comprisesadding a measured amount of a urea-containing sample to a solution ofnon-rate limiting amounts of l-glutamic dehydrogenase, the reduced formof fl-nicotinamide-adenine dinucleotide, and sodium-w ketoglutarate,said solution being buttered to a pH of 7.8-7.9; thereafter, adding anamount of an aqueous glycerol solution of urease sufiicient to releaseammonia from said urea-containing sample at a measurable rateproportional to the concentration of urea in said sample; and measuringthe rate of disappearance of the reduced form of \B-nicotinamide-adeninedinucleotide while maintaining the system at a substantially constanttemperature of 37 C.

8.'A process for the quantitative determination of an enzyme selectedfrom the group consisting of urease, uricase, l-lysine amino acidoxidase, histidine-a-deaminase, l-serine dehydrase, l-threonine aminoacid oxidase, lhomoserine amino acid oxidase, l-cysteine amino acidoxidase, glycine oxidase, l-aspartase, quanine deaminase, monoamineoxidase, d-amino acid oxidase and l-glutaminase which comprises adding ameasured amount of a sample containing said enzyme to a solution oflglutamic dehydrogenase, u-glutarate, the reduced form offi-nicotinamide-adenine dinucleotide and an excess of anitrogen-containing compound selected, correspondingly,

from the group consisting of urea, uric acid, l-lysine, lhis'tidine,l-serine, l-threonine, l-homoserine, l-cysteine, glycine, "l-asparticacid, quanine, aliphatic amines, damino acids'and l-glutamine; andmeasuring the rate of disappearance of the-reduced form ofB-nicotinamideadenine dinucleotide while maintaining the system at aconstant temperature.

9. A process for the quantitative determination of urease whichcomprises adding a measured amount of a enzyme to a solution containinga non-rate limiting amount of compound A and a second enzyme, saidcompound A being reactive With ammonia in a reproducible measurablemanner in the presence of said second enzyme; thereafter, adding anon-rate limiting amount of a nitrogen-containing compound which isreactive with said first enzyme to release ammonia from said sample at ameasurable rate proportional to the concentration of said first enzymein said sample; and measuring the rate of reaction of ammonia with saidcompound A while maintaining the system at a substantially constanttemperature and at a substantially constant pH.

11. A process for the quantitative determination of anitrogen-containing compound capable of releasing ammonia upon enzymaticaction which comprises adding a measured amount of a sample of suchnitrogen-containing compound to a solution having excess amounts of afirst enzyme and com-pound A, said compound A being reactive withammonia in a reproducible measurable manner in the presence of saidfirst enzyme; thereafter, adding a second enzyme that reacts with saidnitrogen-containing compound to release ammonia, the amount of saidsecond enzyme added being sutficient to release ammonia from said samplein an amount that is proportional to the concentration of thenitrogen-containing compound in said sample; and measuring the amount ofammonia that reacts with said compound A.

12. A process for the quantitative determination of anitrogen-containing compound capable of releasing ammonia upon enzymaticaction which Comprises adding a measured amount of a sample of suchnitrogen-containing compound to a solution of non-rate limiting amountsof a first enzyme and compound A, said compound A being reactive withammonia in a reproducible measurable manner in the presence of saidfirst enzyme; adding a second enzyme that reacts with saidnitrogen-containing compound to release ammonia, the amount of saidsecond enzyme added being sufl'icient to release ammonia from saidsample at a measurable rate proportional to the concentration of thenitrogen-containing compound in said sample; and measuring the rate ofreaction of ammonia with said compound A while maintaining the system ata substantially constant temperature and at a substantially constant pH.

13. A process for the quantitative determination of znitrogen-containing compound capable of releasing ammonia upon enzymaticaction which comprises adding 2 measured amount of a sample of suchnitrogen-conraining compound to a solution of non-rate limiting zm'ountsof l-glutamic dehydrogenase, a-ketoglutarate, znd the reduced form ofB-nicotinamide-adenine. dinu- :leotide; adding a second enzyme thatreacts with said nitrogen-contatining compound to release ammonia, thezmount of said second enzyme added being sufiicient to 'elease ammoniafrom said sample at a measurable rate 7r0p0rtional to the concentrationof the nitrogen-containng compound in said sample; and measuring therate of lisappearance the reduced form of fl-nicotinamidezdeninedinucleotide while maintaining the system at a rubstantially constanttemperature and at a substantially :onstant pH.

14. A process as in claim 13 wherein said nitrogen- :ontaining compoundis selected from the group consistng of urea, uric acid, l-lysine,l-histidine, l-serine, lhreonine, l-homoserine, l-cysteine, glycine,l-aspartic tcid, quanine, aliphatic amines, d-amino acids andrlutamz'ne, and said enzyme is, correspondingly, selected rom the groupconsisting of urease, uricase, .l-lysine tmino acid oxidase,histidine-adeaminase, l-serine dehylrase, l-threonine amino acidoxidase, l-homoserine tmino acid oxidase, l-cysteine amino acid oxidase,glycine txidase, l-aspartase, quanine deaminase, monoamine oxilase,d-amino acid oxidase and l-glutaminase.

15. A process for the quantitative determination of anitrogen-containing compound capable of releasing ammonia upon enzymaticaction which comprises adding a measured amount of a sample of suchnitrogen-containing compound to a solution of non-rate limiting amountsof l-alanine dehydrogenase, pyruvate, and the reduced form offl-nicotinamide adenine dinucleotide; adding a second enzyme that reactswith said nitrogen-containing compound to release ammonia, the amount ofsaid second enzyme added being Lsufiicient to release ammonia from saidsample at a measurable rate proportional to the concentration of thenitrogen-containing compound in said sample; and. measuring the rate ofdisappearance of the reduced form of fl-nicotinamide adeninedinucleotide while maintaining the system at a substantially constanttemperature and at a substantially constant pH.

16. A procesis for the quantitative determination of anitrogen-containing compound capable of releasing ammonia upon enzymaticaction which comprises adding a measured amount of a sample of suchnitrogen-containing compound to a solution of non-rate limiting amountsof glyoxylate reductase, glyoxylate, and the reduced form offl-nicotinamide-adenine dinucleotide; adding a second enzyme that reactswith said nitrogen-containing compound to release ammonia, the amount ofsaid second enzyme added being sufficient to release ammonia from saidsample at a measurable rate proportional to the concentration of thenitrogen-containing compound in said sample; and measuring the rate ofdisappearance of the reduced form ,B-nicoatinamideadenine dinucleotidewhile maintaining the system at a substantially constant temperature andat a substantially constant pH.

17. A process for the quantitative determination of urea which comprisesadding a measured amount of a urea-containing sample to a solution ofnon-rate limiting amounts of l-glutamic dehydrogenase, a-ketoglutarateand the reduced form of p-nicotinamide-adenine dinucleotide, saidsolution being buffered to a pH of 6.8-9; adding an amount of a solutionof urease sufficient to release ammonia from said urea-containing sampleat a measurable rate proportional to the concentration of urea in saidsample; and measuring the rate of disappearance of the reduced form offl-nicotinamide-adenine dinucleotide while maintaining the system at asubstantially constant temperature selected from between 15 C. and 50 C.

18. A process for the quantitative determination of urea which,comprise's adding a measured amount of a urea-containing sample to asolution of non-rate limiting amounts of l-glutamic dehydrogenase, thereduced form of fi-nicotinamide-adenine dinucleotide, and sodium-wketoglutarate, said solution being bufiered to a pH of 7.8 7.9; addingan amount of an aqueous glycerol solution of urease sufiicient toreledse ammonia from said ureacontatining sample at a measurable rateproportional to the concentration of urea in said sample; and measuringthe rate of disappearance of the reduced form of fl-nicotinamide-adeninedinucleotide while maintaining the system at a substantially constanttemperature of 37 C.

19. A process for the quantitative determination of a first enzyme whichis reactive with a nitrogen-containing compound, said compound beingcapable of releasing ammonia upon reaction therewith, which comprisesadding a measured amount of a sample containing such first enzyme to asolution containing a non-rate limiting amount of compound A and asecond enzyme, said compound A being reactive with ammonia in areproducible measurable manner in the presence of said second enzyme;adding a non-rate limiting amount of a nitrogencontaining compound whichis reactive with said first enzyme to release ammonia from said sampleat a measurablc rate proportional to the concentration of said firstenzyme in said sample; and measuring the rate of reaction of ammoniawith said compound A while maintaining the system at a substantiallyconstant temperature and at a substantially constant pH.

20. A process for the quantitative determination of anitrogen-contatining compound capable of releasing ammonia uponenzymatic action which comprises adding a measured amount of a sample ofsuch nitrogen-co-ntaining compound to a solution having excess amountsof a first enzyme and compound A, said compound A being reactive withammonia in a reproducible measurable manner in the presence of saidfirst enzyme; adding a second enzyme that reacts with saidnitrogen-containing compound to release ammonia, the amount of saidsecond enzyme added being sujficient to release ammonia from said samplein an amount that is proportional to the concentration of thenitrogen-containing compound in said sample; and measuring the amount ofammonia that reacts with said compound A.

10 References Cited The following references, cited by the Examiner, areof record in the patented file of this patent or the original patent.

UNITED STATES PATENTS 9/1961 Albaum et al. 195--103.5 5/1966 Babson195-1035 OTHER REFERENCES ALVIN E. TAN ENHOLTZ, Primary Examiner U.S.C1. X.R. 195-29

